Project Summary The long-term goal of our laboratory is to determine how a hair cell assembles and operate its mechanically sensitive hair bundle, and to determine how defects in genes responsible for bundle structure and function lead to deafness. In this project, we propose to finally accomplish biochemical purification of the hair cell?s transduction complex, the assembly of proteins?including the transduction channel itself?that mediates the mechanical-to-electrical transduction that is the key event in hearing and balance. In addition, as recent evidence has shown that Ca2+ entry through transduction channels fine-tunes stereocilia length and hence perfects the stereocilia staircase, we will purify the protein complexes that control stereocilia dimensions and determine whether they are regulated by transduction through Ca2+ entry. Finally, we will expand our computational model for tip-link regeneration to include stereocilia length regulation, with the goal of simulating the process of final adjustment of the stereocilia staircase. Predictions of this model will be tested in by examining tip links and stereocilia length in the chick cochlea, a robust experimental system for examining hair- bundle development. This project uses advanced biochemical purification techniques, including a new method for elution from immunoaffinity columns that allows sequential antibody purification steps without the use of harsh conditions. We also continue to pioneer the application of high sensitivity mass spectrometry methods for identifying and quantifying proteins in the inner ear. Even though the number of hair cells is limited in the inner ear and the molecules mediating transduction and stereocilia length regulation are scarce, these sensitive methods allow us to use a biochemical approach to investigate these processes. We expect that this project will determine the molecular composition of the transduction apparatus, including stoichiometry of the constituent proteins, and to define the steps in stereocilia dimension regulation that are controlled by Ca2+. Our computational model for transduction?s fine-tuning of stereocilia length should then set the stage for a future comprehensive model that can describe assembly of the hair bundle and its transduction apparatus.